Methods concerning PPAR delta and antagonists thereof

ABSTRACT

Non-human animals which overexpress PPARd or which express transgenic PPARd are useful as models for inflammatory skin conditions such as psoriasis. Test substances can be screened to assess their suitability for the treatment of inflammatory skin conditions such as psoriasis. Methyl 3-({[2-(methoxy)-4-phenyl]amino}sulfonyl)-2-thiophenecarboxylate can be administered topically for the prevention or treatment of psoriasis.

This application is the U.S. national phase of International ApplicationNo. PCT/GB2009/050967, filed 31 Jul. 2009, which designated the U.S. andclaims priority to Application No. GB 0814094.9, filed 1 Aug. 2008, andGB 0822240.8, filed 5 Dec. 2008; the entire contents of each of whichare hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a non-human animal which is operable tooverexpress peroxisome proliferator-activated receptor delta (PPARδ) oroperable to express transgenic PPARδ, in sufficient amount to displaysymptoms characteristic of an inflammatory skin condition, such aspsoriasis, in the presence of a PPARδ agonist. The invention alsorelates to the use of such an animal as a model for an inflammatory skincondition, such as psoriasis, and methods of using such an animal modelfor identifying a substance for use in the treatment of psoriasis oranother inflammatory skin condition. The invention also relates to theuse of PPARδ antagonists, such as methyl3-({[2-(methoxy)-4-phenyl]amino}sulfonyl)-2-thiophenecarboxylate, forthe treatment or prevention of an inflammatory skin condition, such aspsoriasis.

BACKGROUND TO THE INVENTION

Psoriasis is one of the most common skin diseases, affecting 2-4% of theCaucasian population worldwide. Due to its chronic nature, its oftenseverely disfiguring aspect, and the lack of curative therapies, thedisease causes considerable distress to individual patients and presentsa significant cost burden to healthcare providers.

This complex disease is characterized by alterations in a variety ofdifferent cells.

These alterations include epidermal keratinocyte hyperproliferation andaltered differentiation indicated by focal parakeratosis (cell nuclei instratum corneum). Endothelial cells are also hyperproliferativeresulting in angiogenesis and dilation, and express increased levels ofadhesion molecules. A mixed leukocytic infiltrate is observed which iscomposed of activated T-lymphocytes, that produce inflammatorycytokines, as well as an increased number of dermal mast cells. Inaddition, activated dendritic cells are present which synthesize TNFαand IFNα, that are central to the disease. Intracutaneous secretion ofcytokines is thought to mediate some or all of the tissue alterationswhich are observed in psoriasis. These cytokines include tumor necrosisfactor-α (TNFα) and interleukin-1 (IL-1), vascular endothelial growthfactor/vascular permeability factor (VEGF/VPF) and transforming growthfactor-α (TGFα). Also of particular importance is the induction ofinterleukin-12 (IL12), interleukin 22, and interleukin-23 (IL23).

Research into the pathogenesis of psoriatic skin lesions, and thedevelopment of potential treatments for psoriasis, have been severelyhampered by the lack of appropriate animal models. Several investigatorshave produced transgenic animals in which the increased expression ofcytokines, adhesion molecules, signalling molecules, or other proteinsin the skin results in epithelial hyperproliferation, inflammatoryresponses of the epidermis, or altered differentiation. For example,mouse models which overexpress VEGF-A or STAT3 in the epidermis harbourthe most psoriasis-like phenotype known to date (Sano et al. (2005),“Stat3 links activated keratinocytes and immunocytes required fordevelopment of psoriasis in a novel transgenic mouse model”, Nat. Med.,11, 43-39; and Xia et al. (2003) “Transgenic delivery of VEGF to mouseskin leads to an inflammatory condition resembling human psoriasis”,Blood, 102, 161-168). However, none of these models is based on themodelling of up- or down-regulated molecules, as they occur in psoriaticlesional skin, and therefore may only represent “phenocopies” mimickingpsoriasis. Therefore, using these models to explore novel strategies ofinterventions may not be relevant for the human disease and,accordingly, these models have not been widely used for pre-clinicaldrug testing. Moreover, none of these existing models yield a 100%disease penetrance with a rapid onset of disease, further limiting theiruse in high throughput screening. Finally, human psoriasis flare-ups arecommonly triggered by environmental factors. One of the most important“classical” triggers of psoriasis is the so-called Koebner phenomenonwhich refers to the elicitation of skin pathology by mechanical traumasuch as habitual friction on exposed joints, minor cuts, or surgicalwounds. None of the existing animal models have been shown to exhibit aKoebner phenomenon.

Another known animal model uses human psoriatic skin transplanted ontothe skin of a scid mouse. In these animal models, the transplanted skingrafts reportedly implant with greater than 85% graft survival andcontinue to exhibit psoriatic features for at least six weeks aftertransplantation. However, this animal model is difficult to utilize as ascreening method for therapeutic agents as it requires human skin fortransplantation to generate the model. Thus, there are no prior modelsof psoriasis in which the clinical and histopathological phenotype areknown to develop as the result of recreating the overexpression of agene which is known to be overexpressed in lesional psoriatic skin.

There is, therefore, an unmet need for an animal model which is suitablefor investigations into psoriasis. Any such model should reproduce theclinical and histopathological phenotype of human psoriasis to as largean extent as possible. The model should be useful for research and forscreening test substances which may be useful for the treatment ofpsoriasis. The invention aims to provide such a model and also extendsto animal models which are relevant to other inflammatory skinconditions.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention there is provided amethod of preparing a non-human animal model for an inflammatory skincondition, the method comprising providing a non-human animal which isoperable to overexpress PPARδ, or operable to express transgenic PPARδ,and administering to the non-human animal a dose of PPARδ agonist whichis sufficient to mediate symptoms characteristic of the inflammatoryskin condition.

Peroxisome proliferator-activated receptor delta, PPARδ (also known asPPARβ), is a nuclear receptor protein which functions as a transcriptionfactor and contributes to the regulation of adipogenesis, glucosemetabolism, myogenesis, and macrophage function. PPARδ acts as aregulator of gene transcription upon binding of activating ligands. Wehave found that a non-human animal which overexpresses human PPARδ, orwhich is operable to express transgenic PPARδ, provides an improvedmodel of inflammatory skin conditions, such as psoriasis, when PPARδ isexpressed and a PPARδ agonist is provided. Unlike the models of theprior art, the model of the present invention can mimic the clinical andhistopathologic characteristics, and signalling events of humanpsoriasis. Rather than representing a phenocopy of an inflammatorycondition, such as psoriasis, essential elements of human psoriaticsignalling are re-created in vivo. Further benefits, advantages andapplications of the non-human animal model are discussed below.

The inflammatory skin condition may be psoriasis. However, the non-humananimal is useful as model for a wider range of inflammatory skinconditions. For example, the inflammatory skin condition may be aninflammatory skin disease involving immune disregulation and/or alteredepidermal cell turnover, for example, psoriasis, a parapsoriasisdisease, lichen planus, epidermal congenital nevi, pityriasis rubrapilaris, or eczema. The inflammatory skin condition may be aninflammatory skin condition involving genetic lesions located insignalling pathways related to PPAR signalling. The inflammatory skincondition may be an inflammatory skin condition involving chronicregenerative epidermal reactions, for example, wound healing, epidermaldysplasias, or papilloma-associated epidermal hyperproliferation.

The non-human animal is typically a rodent, for example, a mouse. Incontrast with humans, rodents do not normally express PPARδ in theiradult interfollicular skin. The non-human animal may be operable tooverexpress native PPARδ, for example under the influence of anintroduced promoter. However, the non-human animal may be operable toexpress transgenic PPARδ. Typically, the non-human animal is operable toexpress transgenic humanized PPARδ, such as human PPARδ. In this case,the amount of transgenic PPARδ which is expressed in use typicallyexceeds the amount of PPARδ which is expressed in the corresponding wildtype animal, in at least some tissues, such as the sebaceous glands.

By “operable to overexpress PPARδ” or “operable to express transgenicPPARδ” we include the option that the overexpression of PPARδ, or theexpression of transgenic PPARδ, is inducible, for example, because theoverexpression of PPARδ, or the expression of transgenic PPARδ, areunder the control of an inducible promoter. In this case, expressiondoes not occur, or occurs only at a significantly lower level whentranscription is not induced. The method may therefore include the stepof inducing transcription by administering an agent which has the effectof inducing an inducible promoter. The inducible promoter may, forexample, be the CYP1A promoter in which case overexpression of PPARδ, orthe expression of transgenic PPARδ, may be induced by feedingIndole-3-Carbinol (I3C) to the animal. Nevertheless, with this promoter,sufficient transgenic (typically human) PPARδ to mediate at least somesymptoms characteristic of psoriasis, when activated by a sufficientdose of PPARδ agonist, may be expressed even without induction, inrelevant tissues including the sebaceous glands.

Typically, in order to display symptoms characteristic of theinflammatory skin condition, it is necessary to administer a PPARδagonist, such as the ligand PPARδ activator, GW-501516k,(2-Methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)-methylsulfanyl)phenoxy-aceticacid). The PPARδ agonist may be administered topically.

Thus, the non-human animal may display the symptoms characteristic ofpsoriasis responsive to administration of both an inducer and a PPARδagonist. Alternatively, a non-human animal may be provided whichdisplays the symptoms characteristic of psoriasis responsive toadministration of a PPARδ agonist without a requirement for anadditional inducer. Preferably, the non-human animal displays no or fewof the symptoms characteristic of the inflammatory skin condition in theabsence of at least the PPARδ agonist or, in some embodiments, both aninducer and a PPARδ agonist.

Preferably, the non-human animal develops at least the majority,preferably at least eight, and more preferably at least nine or at leastten of the following symptoms: erythematous skin with loose whitishscales; acanthosis, hyperkeratosis and focal parakeratosis; keratinocytehyperproliferation; changes in keratinocyte differentiation; dermalangiogenesis; infiltration of CD4+ and CD8+ T lymphocytes; dilation ofblood vessels; increased number of dermal mast cells; infiltration ofthe dermis with neutrophils; formation of microabscesses within theepidermis; increased numbers of CD11c+ dendritic cells in the dermis,and changes in cytokine and/or gene expression patterns which correspondto changes in cytokine and/or gene expression patterns which areobserved in the skin of human patients with psoriasis.

Preferably, the non-human animal displays a Koebner phenomenon, that isto say, symptoms characteristic of the inflammatory skin condition aremost pronounced on body regions subjected to mechanical trauma.

The non-human animal should preferably overexpress PPARδ, or expresstransgenic PPARδ, in one or more types of cell, including at leastkeratinocytes, to thereby display symptoms characteristic of psoriasisor another inflammatory skin condition. Preferably, the non-human animaloverexpresses PPARδ, or expresses transgenic PPARδ, in its sebaceousglands. Typically, the non-human animal is operable to overexpressPPARδ, or to express transgenic PPARδ, in at least some types of T-cell.It may be that the non-human animal is operable to overexpress PPARδ, orto express transgenic PPARδ, in both the skin (e.g. keratinocytes) andthe spleen simultaneously. Strong symptoms characteristic of psoriasismay be obtained in such animals. It may be that the non-human animal isoperable to overexpress PPARδ, or to express transgenic PPARδ, in a widerange of types of cell.

According to a second aspect of the present invention, there is provideda method of identifying a substance for use in the treatment of aninflammatory skin condition, the method comprising providing a non-humananimal which is operable to overexpress PPARδ, or operable to expresstransgenic PPARδ, administering a dose of PPARδ agonist which would besufficient to mediate symptoms characteristic of the inflammatory skincondition in the absence of test substance to the non-human animal, ortissues and/or cells derived therefrom, and administering a testsubstance to the non-human animal, or tissues and/or cells derivedtherefrom.

Thus, in the absence of test substance, the non-human animal, or tissuesand/or cells derived therefrom, would display symptoms characteristic ofthe inflammatory skin condition. If, in the presence of the testsubstance, some or all of the symptoms which would be displayed in theabsence of test substance, were reduced or absent compared to a controlanimal to which test substance has not been administered, this would beindicative that the test substance, or a derivative or analogue thereof,would be a candidate for use in the prevention or treatment of theinflammatory skin condition.

The inflammatory skin condition may be psoriasis.

The method may comprise the step of measuring STAT3 phosphorylation(typically Tyr-705 phosphorylation in the case of murine STAT3), or abiological indicator regulated by STAT3 phosphorlyation. We have foundthat STAT3 phosphorylation is regulated by PPARδ. Therefore, thepotential of a test substance to be a candidate for use in theprevention or treatment of the inflammatory skin condition might beinvestigated by monitoring the relationship between STAT3phosphorylation, or a biological indicator regulated by STAT3phosphorylation, and the amount or presence of the test substanceadministered to the non-human animal, or tissues and/or cells derivedtherefrom.

Further preferred and optional features of the second aspect of theinvention correspond to those discussed above in relation to the firstaspect of the invention.

The invention extends in a third aspect to the use of a non-human animalwhich is operable to overexpresses PPARδ, or operable to expresstransgenic PPARδ, in sufficient amount for the non-human animal todisplay the symptoms of an inflammatory skin condition in the presenceof a PPARδ agonist, as an animal model for an inflammatory skincondition. The inflammatory skin condition may be psoriasis.

According to a fourth aspect of the present invention, there is provideda non-human animal, in which at least some types of cell, including atleast keratinocytes, are operable to overexpress PPARδ, or operable toexpress transgenic PPARδ, in sufficient amount for the non-human animalto display the symptoms of an inflammatory skin condition in thepresence of a PPARδ agonist.

According to a fifth aspect of the present invention, there is provideda non-human animal, in which at least cells within the sebaceous glandsof the non-human animal are operable to overexpress PPARδ, or operableto express transgenic PPARδ, in sufficient amount for the non-humananimal to display the symptoms of an inflammatory skin condition in thepresence of a PPARδ agonist.

Preferably, the overexpression of PPARδ, or the expression of transgenicPPARδ is inducible, and the non-human animal does not display thesymptoms of psoriasis except when a PPARδ agonist is present and, ifnecessary, the overexpression of PPARδ is induced.

Preferably, the non-human animal displays symptoms of psoriasis in thepresence of a PPARδ agonist.

Preferably, the non-human animal displays at least eight and morepreferably at least nine or at least ten of the following symptoms inthe presence of a PPARδ agonist: erythematous skin with loose whitishscales; acanthosis, hyperkeratosis and focal parakeratosis; keratinocytehyperproliferation; changes in keratinocyte differentiation; dermalangiogenesis; infiltration of CD4+ and CD8+ T lymphocytes; dilation ofblood vessels; increased number of dermal mast cells; infiltration ofthe dermis with neutrophils; formation of microabscesses within theepidermis; increased numbers of CD11c+ dendritic cells in the dermis,and changes in cytokine and/or gene expression patterns which correspondto changes in cytokine and/or gene expression patterns which areobserved in the skin of human patients with psoriasis

In the presence of a PPARδ agonist the non-human animal preferablydisplays increased phosphorylation of STAT3.

Preferred and optional features of the non-human animal correspond tothose discussed above in relation to the non-human animals use in thefirst and second aspects of the invention.

The invention extends in a sixth aspect to the use of a PPARδ antagonistfor the treatment or prevention of an inflammatory skin condition, forexample psoriasis. The invention also extends to the use of a PPARδantagonist for the manufacture of a medicament for the treatment of aninflammatory skin condition, for example psoriasis. The medicament maycomprise a therapeutically effective amount of the PPARδ antagonist anda pharmaceutically acceptable diluent. Typically, the PPARδ antagonistis applied topically in a therapeutically effective amount. Themedicament is typically suitable for topical application.

The PPARδ antagonist preferably does not significant antagonise PPARα orPPARγ.

The PPARδ antagonist is preferably methyl3-({[2-(methoxy)-4-phenyl]amino}sulfonyl)-2-thiophenecarboxylate, or apharmaceutically acceptable derivative thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

An example embodiment of the invention will now be illustrated withreference to the following Figures:

FIG. 1 is a schematic diagram of the system employed for inducibleexpression and activation of PPARδ in vivo;

FIG. 2( a) illustrates Western blot analyses of 10 μg of total proteinextract of transgenic mice fed I3C or control diet in an experiment inwhich PPARδ expression was induced in mice harbouring CYP1A1-controlledPPARδ by oral administration of indole-3-carbinol (I3C) for seven days(top); and an experiment in which PPARδ was expressed by feeding of I3Cand activated by additional administration of the specific PPARδ ligandGW-501516 (GW) for 25 days (bottom); FIG. 2( b) is a close-up photographof the macroscopic phenotype of mice receiving both I3C and GW-501516administration; FIG. 2( c) is a photograph of standard H&E histologystains prepared from abdominal skin of the animals shown in FIG. 2( a);

FIG. 3 illustrates experimental results from arising from the inducibleexpression of PPARδ in mouse epidermis. FIG. 3( a) Cis-regulatoryelements in the rat CYP1A1 promoter used to drive inducible expressionof human PPARδ. Upper panel: Map of the sebaceous-specific G/C-boxelement and the AHR-responsive DXE/XRE cluster in the cyp1A1 promoter aswell as the human human K5 promoter. Bottom panel: ClustalW alignment ofpromoters identified by a BLAST search using the 20 bp G/C element ofthe Cyp1A1 promoter. All of the genes shown were found in the top 10%percentile of all transcripts expressed in human sebaceous glands(GDS3215 at the NCBI GEO website www.ncbi.nlm.nih.gov/geo/). FIG. 3( b)illustrates immunohistochemistry using anti-PPARδ of mice transgenic forhuman PPARδ driven by the rat CYP1A promoter (PPARδ TG), as well as wildtype mice. Magnification 200×; FIG. 3( c) illustratesimmunohistochemistry of abdominal skin samples from PPARδ transgenicmice taken at the indicated times after initiation of PPARδ activationmediated by administration of the synthetic ligand GW501516 in the chow;FIG. 3( d) illustrates PPARδ immunohistochemistry 48 h after topicalapplication of indole-3-carbinole (I3C) to the skin of PPARδ transgenicmice at 200× (left) and 400× (right) magnification;

FIG. 4 illustrates immune activation in PPAR(-mediated skin disease. (a)immunohistochemistry for CD4, CD8, CD11c, and CD31 (Pecam 31) of skinfrom PPAR(-transgenic mice maintained in the absence (top) or presenceof GW501516. Magnification 200×, (b) flow cytometry analysis showingintracellular FACS-staining for IFN (and IL17 of skin cells (gated forCD4) from wild type and PPAR (transgenic mice maintained in the presenceor absence of GW501516, respectively. Numbers in quadrants indicatefrequency of positive cells, (c) frequency of CD4+IL17+ of IL17+ cells(expressed as percent of all CD4+ gated cells) in PPAR (transgenic andC57B1/6 wild type mice maintained in the presence or absence of GW501516(n=4 per group), as determined by flow cytometry. *p<0.01; **p<0.001,(d) frequency of CD4+IL17+ Th17 cells (left y-axis, black columns) andratio of IL17+ and IFN(+ cell frequencies (right y-axis, grey columns)in the skin of PPAR (mice maintained in the absence or presence ofGW501516 with or without i.p. injection of anti-TNF(, or (IL12/23p40(n=4, see Methods), (e) disease severity, expressed as mean±s.d.,assessed by the degree of erythema, thickening, scaling, and hair loss(see Methods, representative photographs of mice on day 19 postinduction are shown in FIG. S6) in PPAR (transgenic miceGW501516—containing chow with or without additional intraperitonealinjection of anti-TNF (or (IL12/23p40 (anti-IL12). *p<0.01, **p<0.001(treatment vs. control);

FIG. 5. Skin phenotype in PPARδ transgenic mice twenty days afterbeginning of GW501516 (GW) administration. (FIGS. 5 a-5 c) Grossmorphology, (FIGS. 5 d-5 e) H&E histology of control mice not treatedwith GW (5 d), or fourteen days after induction (5 e-5 g). Magnification200× (5 d,5 e) or 400× (5 f). (5 g) Immunostaining for Ki67 of skin fromPPARδ transgenic mice maintained in the absence (left) or presence(right) of GW. Magnification 200×. (5 h) Induction of skin disease bytopical application of either 0.3% of indole-3-carbinole (I3C, left) orI3C plus 0.3% GW501516 once daily to shaved abdominal skin. Grossmacroscopic phenotype (top) and H&E histology of treated skin (bottom)was documented 10 days after beginning of treatment; and

FIG. 6. Activation of STAT3 by PPARδ, (a) Western blot of whole skinsamples from two GW501516-treated (GW) and two control PPAR transgenicmice, respectively, probed with anti phospho-STAT3 (top) and anti-STAT3(bottom) along with anti-GAPDH loading controls (top-band of the STAT3doublet represents STAT3α, bottom-band STAT3β, respectively), (b)immunofluorescence with anti phospho-STAT3 of skin from GW-treated andcontrol PPARδ mouse in the absence (left) or presence (right) ofcompetition with antigenic peptide. White dashed line marksdermo-epidermal boundary (not marked in control samples where epidermisis only two cell layer-thick), all samples at 400× magnification. Theinset in the upper left panel (1000×) shows counter-stain with DAPI toverify nuclear localization of phospho-STAT3. (c) gross appearance ofGW-fed PPARδ mouse (left) or mouse concurrently IP-injected with WP1066(right), as detailed in Methods, (d) H&E histology of skin samples frommice shown in (d) at 200× magnification, (e) fold change of genesrepressed by activated STAT3 (dark grey columns, data taken from (Daueret al. (2005), “Stat3 regulates genes common to both wound healing andcancer.” Oncogene, 24, 3397-3408.) in GW501516-fed vs. control PPAR(transgenic mice (white), lesional vs. non-lesional skin from psoriasispatients in the GSE14905 (black), and the GAIN (light grey) datasets,respectively. * denotes genes that did not meet a p-value<0.01 and arethus were formally excluded from cluster IV (table S2), (f) Taqman-basedqPCR of IFI27 from whole skin of untreated, GW-fed, andGW-fed+WP1066-injected PPAR (mice, respectively. *p<0.05.

Experimental

Mice were generated which are inducible to overexpress human PPARδ inthe skin and a wide range of other tissues and organs, including thespleen, under the control of the CYP1A1 promoter. The CYP1A1dioxin-responsive promoter was initially chosen because it is known tobe transactivated by the aryl hydrocarbon receptor (AHR) upon binding ofspecific AHR agonist ligands. (Campbell et al., Regulation of the CYP1A1promoter in transgenic mice: an exquisitely sensitive on-off system forcell specific gene regulation, J. Cell Science, 1996, 109 (Pt. 11),2619-2625).

The mice were generated by cloning full-length human PPARδ downstream ofthe human CYP1A1 promoter. Plasmids encoding human PPARδ were preparedby the following protocol. The coding sequences of PPARδ, were amplifiedusing primers PRMG15 (5′-CTAGTCTAGAATGGAGCAGCCACAGGAGGAAGC-3′) and PRMG3 (5′-CTAGTCTAGATTAGTACATGTCCTTGTAGATCTCCTG-3′), respectively(XbaI-sites underlined, ATG start codon in bold). The PCR products werecleaved with XbaI and cloned in plasmid pUHD10-3 (M. Gossen,unpublished, Genbank accession number U89931) creating pMGD7 (PPARδ).The integrity of the inserts were confirmed by sequencing and cleavedout using BamHI and ligated into plasmid pAHIR1-β-gal (Campbell, 1996}cleaved with BglII, resulting in the plasmid pMGD72 (PPARδ). The properorientations of the inserts were confirmed by restriction endonucleaseanalysis and sequencing. Transgenic mice were generated bymicroinjection of the expression unit (NotI fragment) of the plasmidpMGD72 into pro-nuclei of C57BL/J6×CBA F1 fertilized eggs. Mice weremaintained under standard animal house conditions.

In a first study, indole-3-carbinole (I3C) introduced into the chow at afinal concentration of 0.5% (w/w), for one week, was used to induceexpression of PPARδ. The resulting translated protein wasconformationally activated by the specific PPARδ ligand GW-501516(2-Methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)-methylsulfanyl)phenoxy-aceticacid), added in powdered form to the chow at a concentration of 0.003%(w/w) for an additional 16 days. This activation mechanism isillustrated in FIG. 1. Thus, PPARδ can be transcriptionally activatedand conformationally activated by concurrent feeding with I3C andGW-501516.

The mice were prepared for the purposes of studies concerning a widerange of cancer phenotypes and liver fat accumulation. When PPARδ wasinduced upon feeding of I3C, strong induction of PPARδ expression wasnoted both in the skin and in lymph notes (FIG. 2 a). When we activatedthe overexpressed PPARδ by additional administration of the specificPPARδ agonist ligand GW-501516, a dramatic skin phenotype was observedafter 25 days, including widespread scaling, skin thickening, andinflammation (FIG. 2 a, bottom and FIG. 2 b). Histologically, the skinof mice harbouring activated PPARδ showed greatly expanded epidermallayers, as well as dermal infiltration of inflammatory cells (FIG. 2 c).This phenotype is similar to that seen in human psoriasis. Rodents, suchas mice, do not normally express PPARδ in their skin and in controlexperiments in which GW-501516 was administered to unmodified mice, nopsoriatic symptoms were observed.

Although upregulation of PPARδ was known to be associated withpsoriasis, the expression and/or activation of many proteins is known tobe upregulated in psoriasis. The striking similarity of the resultingphenotype to human psoriasis indicates that the transgenic mice aresuitable as animal models for investigations into psoriasis. Thesymptoms displayed by the transgenic mice included the followingclinical and histopathological features which are also characteristic ofhuman psoriasis: dermal and epidermal infiltration of T lymphocytes inassociation with erythematous skin with loose whitish scales;acanthosis, hyperkeratosis and focal parakeratosis; keratinocytehyperproliferation; changes in epidermal differentiation markers;increased numbers of CD11c+ dendritic cells; prominent dermalangiogenesis and dilation of blood vessels; increased number of dermalmast cells, and infiltration of neutrophils. The skin phenotype istypically visible within a week and full blown within around twenty daysin all mice, thus making it feasible to study its response to a varietyof manipulations, or to employ the mice in high throughput screens forcandidate therapeutic entities.

It was discovered that induction with I3C was not required for thesymptoms of psoriasis to be displayed. In addition to a well-documentedDXE/XRE cluster conferring responsivity to AhR activation (Robertson etal. (1994) “Aryl hydrocarbon-induced interactions at multiple DNAelements of diverse sequence-a multicomponent mechanism for activationof cytochrome P4501A1 (CYP1A1) gene transcription”, Nucleic acidsresearch, 22, 1741-1749). the Cyp1A1 enhancer contains a cis-actingelement conserved in the keratin 5 promoter (FIG. 3 a, upper panel).This element, termed K5 4.2-5′, directs AhR-independent, highly specificconstitutive expression in sebaceous glands (Kaufman et al. (2002),“Dissection of a complex enhancer element: maintenance of keratinocytespecificity but loss of differentiations specificity. Molecular andCellular Biology., 22, 4293-4308.) Thus, the rat CYP1A1 promoter alsodrives Ahr-independent sebaceous gland-specific expression of an EGFPreporter in mice (Rowe et al. (2008), “Illuminating role of CYP1A inskin function”, The Journal of Investigative Dermatology, 128, 110-124.We identified the K5 4.2-5′ element as a universal “sebaceous box”present in the promoters of genes belonging to the top 10% of allexpressed genes in sebaceous glands (FIG. 3 a, bottom). Accordingly,cloning human PPARδ downstream of the same promoter efficientlyconferred high constitutive sebaceous-specific expression of PPARδ (FIG.3 b). We hypothesized that activation of PPARδ in the sebaceous glandsshould elicit secondary expression of PPARδ in the upper epidermis viathe DXE/XRE cluster in the CYP1A1 enhancer since PPARδ stimulatessebocyte differentiation (Michalik and Wahli (2007), “Peroxisomeproliferator-activated receptors (PPARs) in skin health, repair anddisease. Biochimica et biophysica acta, 1771, 991-998; Rosenfield et al.(1999), “Rat preputial sebocyte differentiation involves peroxisomeproliferator-activated receptors”, J. Invest. Dermatol., 112, 226-232)and delivery of sebum to the skin (Trivedi et al. (2006), “Peroxisomeproliferator-activated receptors increase human sebum production”, TheJournal of Investigative Dermatology, 126, 2002-2009) which, in turn,triggers epidermal delivery of lipoxygenase-products serving asendogenous AhR ligands such as LXA4 or 5,6-DiHETE (Chiaro et al. (2008),“Leukotriene A4 metabolites are endogenous ligands for the Ah receptor”,Biochemistry 47, 8445-8455; Machado et al. (2006), “Anti-inflammatoryactions of lipoxin A4 and aspirin-trigerred lipoxin are SOCS-2dependent”, Nature Medicine, 12, 330-334).

Indeed, in a second study, upon activation of PPARδ using the selectiveligand GW501516, without I3C, we observed strong expression of PPARδ inthe epidermis within 48 h (FIG. 3 c). At later time points, PPARδ wasstrongly expressed in cells of the spinous layer PPARδ expression wasepidermis-specific and did not occur in dermal fibroblasts, endothelia,or skin-associated T cells (see below). PPARδ expression was alsoinducible by topical application of the AhR ligand indole-3-carbinole tothe skin (I3C, FIG. 3 d), thereby confirming that epidermal induction istriggered by the AhR-responsive element in the Cyp1A1 promoter. The neteffect is a tightly controlled inducible expression of PPARδ insuprabasal mouse epidermis which is comparable to that observed in humanpsoriasis lesions.

As early as seven days after initiation of PPARδ-activation by GW501516(GW), scaling, erythema formation, and skin thickening was notable inall PPARδ transgenic mice (FIG. 4 a-c). Hyperkeratosis and concomitanthair loss was maximal in regions subjected to mechanical friction, suchas abdomen (FIG. 4 b, S3), the paws (FIG. 4 a), or the chin (FIG. S2).While hyperkeratotic plaques were also noted on the back in some mice(FIG. 4 c) changes on the dorsal skin were mostly limited to scaling(FIG. S2). Thus, the extent of clinical changes displayed a pronouncedmechanical trigger effect similar to the isomorphic trigger effect seenin psoriasis. Histology showed epidermal thickening (FIG. 4 e), dilationof dermal vessels, and a lymphocytic dermal infiltrate. Of note, incontrast to psoriasis, the granular layer was prominent (FIG. 4 f),consistent with the known effect of PPARδ on epidermal differentiation.Moreover, K67 staining demonstrated massive hyperproliferation in thebasal layer of the epidermis (FIG. 4 g). Importantly, these changes weremediated solely by ligand-mediated activation of PPARδ, but not by AhRactivation since prolonged administration of a chow containing a veryhigh I3C concentration (0.5% w/w) caused no skin changes in the absenceof PPARδ activation (not shown). Likewise, topical administration of I3Cto the skin was unable to elicit a phenotype but was effectivelyreplicated by additional topical addition of GW501516 (FIG. 4 h). Thus,the skin phenotype in PPARδ transgenic mice is mediated by activation ofPPARδ in suprabasal keratinocytes.

In contrast to known animal models, the model of the present inventionmimics the clinical and histopathologic characteristics of inflammatoryskin conditions, such as human psoriasis, within seven days, offers ahighly controllable and rapid disease onset by dose-controlledadministration of specific transgene activators, and occurs in virtuallyall experimental animals. Moreover, the disease occurs as a result offorced upregulation of a protein as it occurs in human psoriasispatients, irrespective of their individual genetic risk profile, thusreplicating a pathological signalling event operative in the humandisease. Therefore, the model does not merely represent a phenocopy ofpsoriasis, but re-creates elements of human psoriatic signalling invivo. Experiments indicate that the model shows the same dynamicplasticity to environmental factors as human psoriasis, for example, aclassical Koebner phenomenon is observed leading to highly aggravateddisease activity at areas of mechanical friction. Accordingly, the modeldisclosed herein is useful for studying the immunopathogenesis of humanpsoriasis, and for evaluating therapeutic agents for ameliorating orpreventing inflammatory skin conditions, such as psoriasis. Inparticular, the model is useful for studying the properties of theepidermis and dermis as a metabolic organ, which have not been wellstudied to date.

The model is useful for studying the direct or indirect effects of testsubstances, or screening test substances (such as small chemicalentities or biologics) to identify substances which may be useful forthe treatment or prevention of psoriasis or other inflammatory skinconditions. A test substance can be administered to the mouse by anappropriate method, such as feeding, gavage, intravenous injection,retro-orbital injection, intra-arterial injection, intra-peritonealinjection or subcutaneous injection. The test substance may beadministered to the mouse after transcriptional and conformationalactivation of PPARδ to investigate the effects of the test substance onpsoriatic symptoms which have already developed, after transcriptionactivation but before conformational activation, or before eithertranscriptional or conformational activation to investigate whether andhow the test substance affects or prevents the development of psoriaticsymptoms.

In order to determine the efficacy of a test substance as a candidatefor the prevention or treatment of psoriasis, or another inflammatoryskin condition, the presence of some or all of the following symptomscan be measured on a quantitative or qualitative basis and compared witha suitable control: erythematous skin with loose whitish scales;acanthosis, hyperkeratosis and focal parakeratosis; keratinocytehyperproliferation; changes in keratinocyte differentiation; dermalangiogenesis; infiltration of CD4+ and CD8+ T lymphocytes; dilation ofblood vessels; increased number of dermal mast cells; infiltration ofthe dermis with neutrophils; formation of microabscesses within theepidermis; increased numbers of CD11c+ dendritic cells in the dermis,and changes in cytokine and/or gene expression patterns which correspondto changes in cytokine and/or gene expression patterns which areobserved in the skin of human patients with psoriasis.

A test substance may be considered to be efficacious if it prevents orreduces the extent of one or more of the above symptoms or, inexperiments where the test substance is administered before psoriaticsymptoms have developed, a test substance may be considered to beefficacious if it delays the onset of one or more of the above symptoms.

The efficacy of a test substance may be established by visual inspectionof symptoms or by measuring the amount, activation or properties ofcells which are potentially implicated in the response to PPARδactivation, for example, the amount and activation of regulatoryT-cells, dendritic cells, natural killer cells, B-cells, andmacrophages. In order to assess the efficacy of a test substance, it maybe relevant to measure the amount of antigens such as CD4, CD25, Foxp3,IL17, CD69, B220, DX5, F4/08 and to quantify the production of cellssuch as CD8 and CD4 cells, or to measure keratinocytehyperproliferation.

Furthermore, we have found that PPARδ regulates phosphorylayion ofsignal transducer and activator of transcription 3 (STAT3). STAT3 ishyperphosphorylated in psoriasis and is sufficient to induce apsoriasis-like phenotype in vivo. We found that Tyr-705 phosphorylationof STAT3 was markedly increased in lesional skin of PPARδ transgenicmice (FIG. 6 a) and localized to the nuclei of suprabasal cells in theepidermis (FIG. 6 b). Inhibition of STAT3 effectively prevented theonset of disease, demonstrating the central role of this pathway (FIG. 6c,d). Strikingly, the single group of genes upregulated in psoriasiswhile downregulated in PPARδ mice, the interferon response genes havepreviously been shown to be repressed by STAT3 (FIG. 6 e, dark shadedcolumns), as part of what has been termed the “anti-inflammatoryresponse” (Murray, P. J. (2006), “STAT3-mediated anti-inflammatorysignaling.” Biochemical society transactions, 34, 1028-1031. Indeed,inhibition of STAT3 signalling partially reversed the down-regulation ofone of the most repressed transcripts, IFI27 (FIG. 6 f), indicating thatthe inhibition of IFN signalling in PPARδ transgenic mice is mediated inpart by STAT3.

Accordingly, the efficacy of a test substance may be assessed byassaying STAT3 phosphorylation, or another biological indicator known tobe affected by STAT3 phosphorylation status. This may enable a morerapid initial screen to be carried out than would be the case if it wasnecessary to wait for a wide range of psoriatic symptoms to beexpressed. STAT3 phosphorylation may be assayed using, for example, anantibody which has an affinity for STAT3 which depends on Tyr-705phosphorylation status.

Examples of suitable test substances include PPARδ antagonists, such ascyclosporin A or CTLA4-IG, as well as libraries of small molecules,peptides, peptidomimetics, antibodies and antibody fragments.

One test substance which may be employed for the prevention or treatmentof psoriasis, or another inflammatory skin condition, is methyl3-({[2-(methoxy)-4-phenyl]amino}sulfonyl)-2-thiophenecarboxylate, knownas GSK 0660, or a pharmaceutically acceptable derivative or saltthereof. GSK 0660 is described in Shearer B. G. et al., (2008),“Identification and Characterization of a Selective PeroxisomeProliferator-Activated Receptor β/δ (NR1C2) Antagonist”, MolecularEndocrinology, 22(2): 523-529, the contents of which are incorporatedherein by virtue of this reference. GSK 0660 is available fromSigma-Aldrich (St. Louis, USA), CAS No. 1014691-01-2. GSK 0660 is ahighly selective inhibitor of PPARδ. Although GSK 0660 lacks in vivobioavailability, it may be applied topically for the treatment ofpsoriasis, or other inflammatory skin conditions. Indeed, the poor invivo bioavailability may be advantageous as effects will be confined tothe skin, reducing or avoiding side effects in other organs.

Tissue or cells from the mouse model may also be useful in research.Tissue or cells may be removed from the mouse model after the inductionof psoriatic symptoms. Tissue or cells may be removed beforetranscriptional activation and then PPARδ within the tissue or cells canbe transcriptionally and then conformationally activated. Tissue orcells may be removed after transcriptional activation but beforeconformational activation and then PPARδ within the tissue or cells canbe conformationally activated. The difference between the physiologicalresponse of isolated tissue or cells types to transcription and/orconformational activation, and their response in vivo can be compared toinvestigate components of the immune system which are relevant toinduction of the skin pathology. Tissue or cells from the mouse modelmay be investigated in vitro, or transplanted to a host organism, suchas a scid mouse.

The model can also be employed to study the effect of PPARδ activationin specific cell subsets. Specific cell types such as subsets ofdendritic cells or T lymphocyte subsets, for example generated by invitro generation, can be transferred to non-transgenic congenicrecipient mice in order to evaluate the contribution of PPARδ activationexclusively in the transferred cell subset on the development of aninflammatory skin condition, such as psoriasis, in vivo. The same canalso, for example, be performed by grafting skin from PPARδ transgenicanimals onto non-transgenic recipient skin.

Instead of employing the CYP1A1 promoter, the overexpression of PPARδ,or the expression of transgenic PPARδ, may be effected using alternativepromoters which control expression in relevant tissues. Constitutive andinducible promoters can each be considered. A keratinocyte specificpromoter (for example, Keratin 5 or Keratin 14 promoter) may be employedto cause PPARδ to be expressed specifically in keratinocytes. A Th-17specific promoter (for example, the IL23-R promoter) might also beemployed. These may lead to an improved model. Alternatively, it may beessential for PPARδ to be expressed at a sufficiently high level in arange of tissues including, for example, the spleen, in order forsymptoms to be displayed. Cell types which may be important or essentialfor the displayed phenotype include regulator T-cells, so-called Th17cells, dendritic cells, and natural killer (NK) cells. The promotershould preferably be selected to avoid strong constitutive expression intissues where the transgene may have substantial toxic effects, e.g. theliver.

Indeed, experiments with an animal model according to the exampleembodiment have demonstrated strong similarities in gene dysfunctionbetween these mice and human psoriasis. Th17 cells, which causeautoimmune activity in human psoriasis have been found to be upregulatedin the mice and a subsequent downregulation of these cells has beenfound to be correlated with a reduction in symptoms, indicating theirimportance.

Thus, in summary, the invention provides an animal model useful forresearch into inflammatory skin conditions, including psoriasis, basedupon the recognition that over activation of PPARδ in the skin ofanimals such as rodents induces an inflammatory skin condition thatclosely resembles human psoriasis. Although other animal models havebeen reported for human psoriasis and other inflammatory skinconditions, the animal model of the invention represents the first invivo experimental system which has induced the expression of a geneknown to be expressed in human psoriatic skin but is not expressed inmouse skin (humanized mouse model), can be induced in virtually allexperimental animals within four weeks, and does not require the use ofhuman skin or cross implantation of lymphocytes for model generation.Thus, the animal model disclosed herein is not a disease-affected skintransplantation animal model or a T cell transplantation model of aninflammatory skin disease and has significant advantages over theseknown models.

Further variations and modifications may be made within the scope of theinvention herein disclosed.

The invention claimed is:
 1. A method of preparing a mouse model for an inflammatory skin condition, the method comprising providing a mouse which expresses transgenic Peroxisome Proliferator-Activated Receptor delta (PPARδ) and administering to the mouse a dose of PPARδ agonist which is sufficient to mediate symptoms characteristic of the inflammatory skin condition.
 2. A method as claimed in claim 1, wherein the mouse expresses transgenic human PPARδ.
 3. A method as claimed in claim 1, wherein the expression of transgenic PPARδ is inducible and the method comprises the step of inducing the expression of transgenic PPARδ.
 4. A method as claimed in claim 1, for preparing a mouse model for psoriasis, wherein the mouse develops symptoms characteristic of psoriasis responsive to administration of the dose of PPARδ agonist.
 5. A method as claimed in claim 1, wherein the mouse develops at least eight of the following symptoms: erythematous skin with loose whitish scales; acanthosis, hyperkeratosis and focal parakeratosis; keratinocyte hyperproliferation; changes in keratinocyte differentiation; dermal angiogenesis; infiltration of CD4+ and CD8+ T lymphocytes; dilation of blood vessels; increased number of dermal mast cells; infiltration of the dermis with neutrophils; formation of microabscesses within the epidermis; increased numbers of CD11c+ dendritic cells in the dermis, changes in cytokine and/or gene expression patterns which correspond to changes in cytokine and/or gene expression patterns which are observed in the skin of human patients with psoriasis.
 6. A method as claimed in claim 1, wherein the mouse displays a Koebner phenomenon.
 7. A method as claimed in claim 1, wherein the mouse expresses transgenic PPARδ in one or more types of cell, including at least keratinocytes, to thereby display symptoms characteristic of psoriasis.
 8. A method as claimed in claim 7, wherein the mouse expresses transgenic PPARδ in at least some types of T cell, to thereby display symptoms characteristic of psoriasis.
 9. A method of identifying a substance for use in the treatment of an inflammatory skin condition, the method comprising providing a mouse which expresses transgenic PPARδ, administering a dose of PPARδ agonist which would be sufficient to mediate symptoms characteristic of the inflammatory skin condition in the absence of test substance to the mouse, and administering a test substance to the mouse, or tissues and/or cells derived therefrom.
 10. A method as claimed in claim 9, wherein the mouse expresses transgenic human PPARδ.
 11. A method as claimed in claim 9, wherein the mouse is of a species, the wild type of which does not express significant amounts of PPARδ in the skin.
 12. A method as claimed in claim 9, for identifying a substance for use in the treatment of psoriasis wherein, in the absence of test substance, the mouse would develop symptoms characteristic of psoriasis.
 13. A method as claimed in claim 9, wherein, in the absence of test substance, the mouse would develop at least eight of the following symptoms: erythematous skin with loose whitish scales; acanthosis, hyperkeratosis and focal parakeratosis; keratinocyte hyperproliferation; changes in keratinocyte differentiation; dermal angiogenesis; infiltration of CD4+ and CD8+ T lymphocytes; dilation of blood vessels; increased number of dermal mast cells; infiltration of the dermis with neutrophils; formation of microabscesses within the epidermis; increased numbers of CD11c+ dendritic cells in the dermis, and changes in cytokine and/or gene expression patterns which correspond to changes in cytokine and/or gene expression patterns which are observed in the skin of human patients with psoriasis.
 14. A method as claimed in claim 9, wherein, in the absence of test substance, the mouse displays a Koebner phenomenon.
 15. A method as claimed in claim 9, comprising the step of assaying the phosphorylation of STAT3, or measuring another biological indicator linked to the activation state of STAT3.
 16. A method as claimed in claim 9, wherein symptoms characteristic of the inflammatory skin condition in the mouse, or the tissue and/or cells derived from a said mouse, are compared with a control to which the test substance is not administered, or is administered in a different amount.
 17. A method as claimed in claim 9, wherein the mouse expresses transgenic PPARδ in one or more types of cell, including at least keratinocytes or within the sebaceous glands.
 18. A method as claimed in claim 17, wherein the mouse expresses transgenic PPARδ in at least some types of T-cell.
 19. A transgenic mouse, in which at least some types of cell, including at least keratinocytes, express transgenic PPARδ in sufficient amount for the mouse to display symptoms characteristic of an inflammatory skin condition in the presence of a PPARδ agonist.
 20. A transgenic mouse as claimed in claim 19, wherein the expression of transgenic PPARδ is inducible, and wherein the mouse does not display symptoms characteristic of psoriasis except when the expression of transgenic PPARδ is induced and a PPARδ agonist is present.
 21. A transgenic mouse as claimed in claim 20, which displays symptoms characteristic of psoriasis in the presence of a PPARδ agonist.
 22. A transgenic mouse as claimed in claim 21, which displays at least eight of the following symptoms, in the presence of a PPARδ agonist: erythematous skin with loose whitish scales; acanthosis, hyperkeratosis and focal parakeratosis; keratinocyte hyperproliferation; changes in keratinocyte differentiation; dermal angiogenesis; infiltration of CD4+ and CD8+ T lymphocytes; dilation of blood vessels; increased number of dermal mast cells; infiltration of the dermis with neutrophils; formation of microabscesses within the epidermis; increased numbers of CD11c+ dendritic cells in the dermis, and changes in cytokine and/or gene expression patterns which correspond to changes in cytokine and/or gene expression patterns which are observed in the skin of human patients with psoriasis. 